Accomplishments 1: Assess which macaque model better predicts or recapitulates the efficacy of HIV vaccine candidates in humans. The SIVmac251 model has predicted and recapitulated RV144: In 2005, a collaborative study in neonates predicted the results of RV144. More recently we recapitulated the efficacy of the RV144 vaccine regimen in young (2-3 years) male and female macaques. The latter study has provided more value than just the evaluation of vaccine efficacy. Our integrated study of RNA expression in PBMCs and exosomes together with comprehensive analyses of innate and adaptive immune responses in blood, lymph nodes, and mucosa uncovered unexpected correlates of risk of virus acquisition such as mucosal innate lymphoid cells (ILCs) and the expression of 12 genes, ten of which are part of the RAS pathway. The usefulness of this model was validated by similar observations in another protective vaccine regimen that I developed in collaboration with Dr. Strbo and the late Dr. Eckhart Podack of Miami University. This approach was the subject of a joint patent. We have also performed a confirmatory study entirely supported by the Division of AIDS, NIAID, in other animal facilities wherein we compared the immunogenicity and efficacy of the ALVAC and NYVAC SIV-based vaccine regimens. The ALVAC-SIV based vaccine was also protective in Chinese macaques, whereas the more immunogenic NYVAC-based SIV recombinant vaccine was not. Of note, the NYVAC-based vaccine did not induce an early burst of IL-1B as was the case in the non-protective Ad26 prime regimen. Will the SIVmac251 model predict the results of HVTN-702? We tested the efficacy of the ALVAC-based SIV vaccines in the SIVmac251 model using the alum in parallel with the MF59 adjuvant in the gp120 boost. To our surprise, the MF59 based regimen in the SIV model was not efficacious, despite its higher immunogenicity. While this observation stirred debate in the scientific community, our findings did not influence the decision by the P5 Partnership to use MF59 as an adjuvant in the HVTN-702 study in South Africa. Instead, logistical and political factors likely influenced the decision to proceed as originally planned. Given the current uncertainty of how accurately the SIVmac251 animal model will predict human results, our study will provide crucial information to define the model's utility since we observed no efficacy in macaques. The preliminary analysis of futility in HVTN-702 will be made public in 2018 and will address this issue. SHIV-clade C Tier 1 in female Chinese rhesus macaques: In another study supported mostly by NIAID, macaques were immunized with the identical ALVAC-HIV-based vaccines used in HVTN702, combined with either the MF59 or the alum (Novartis) in the gp120 boost, and challenged with a SHIV clade C Tier 1 virus. The MF59 based regimen was protective, whereas the alumN was not. Protection was associated with neutralizing antibodies to the SHIV clade C Tier 1 virus. In its current state, I don't believe that this SHIV model will predict the results of HVTN-702 because only 1% of HIV clade C viruses in South Africa are sensitive to neutralization. However, the interim analysis of HVTN-702 for futility will further our knowledge of animal model predictability and will guide the choice and refinement of faithful animal models for the development of fully effective vaccines for HIV. 2. HIV vaccine candidate activation of hypoxia and inflammasome in CD14+ monocytes is associated with a decreased risk of SIVmac251 acquisition. This study demonstrated that CD14+ and CD16+ monocytes have a role in the risk of SIVmac251 acquisition and has contributed to the mechanistic insight on the role of IL-1B and IL-10 in the DNA/ALVAC/alum vaccine platform. We compared ALVAC-SIV, DNA-SIV, and Ad26-SIV prime modalities in combination with two ALVAC-SIV + gp120 protein boosts in young male macaques. We used the intramuscular DNA-SIV prime with the intent of increasingCD4+ T cells and antibody responses since we demonstrated in prior work that the CD4+ T cells elicited by the DNA prime influence the anti-envelope titers. The Ad26-SIV prime was chosen with the intent of increasing mucosal antibodies to Env and CD8+ T cell responses. The DNA prime protected against infection in the DNA prime regimen, and VE correlated with hypoxia and inflammasome activation in classical CD14+ CD16negative monocytes that in turn directly correlated with the gut-homing CCR5negative Th2 cell responses and with mucosal NKp44+ cells and IgG to V2. In the same group, the frequency of differentiated CD16+ monocytes correlated with reduced vaccine efficacy via STAT3 activation. The Ad26 prime regimen did not protect against SIV acquisition, despite its higher immunogenicity, and it was associated with a decrease in total CD14+ DR+ monocytes in blood, accumulation of de novo differentiated CX3CR1+ CD163+ macrophages in lymph nodes, mucosal inflammation, and skewing of CD4+ T cells toward Th17 cells in the rectal mucosa and lungs. Th17 cells are preferentially infected by HIV/SIV and negatively regulate NKp44+ cells. It is possible that vaccine-generated target cells for virus infection, may have decreased the efficacy of the Ad26 prime regimen, as was observed in other vaccine studies in macaques and humans. 3. A simplified vaccine regimen based on DNA/ALVAC-SIV / ALVAC-SIV + gp120 / alum affords 69% vaccine efficacy. With the intent of confirming the importance of monocyte-mediated innate immunity and investigating whether the DNA/ALVAC/gp120/alum approach was also effective in female macaques, we designed a study in young and old females. We tested a simplified version of the DNA prime ALVAC/gp120/alum vaccine approach using two intramuscular DNA primes (weeks 0 and 4), one ALVAC-SIV boost (week 8), and a single final boost with the combination ALVAC-SIV and gp120 in alum at week 13. Challenge exposure was started at week 17 and continued for 11 weeks. We found that this vaccine had 69% VE in young female macaques (average age 2.7 years), but failed to protect old female macaques (average age 7.5 years). Protection in young females correlated with the frequency of CD14+ classical monocytes, confirming the results of the previous study in young males. In old vaccinated females, however, we observed a correlation with the plasma level of IL-6 and IL-8 and increased virus acquisition. These data further support a role for monocytes in protection of both female and male macaques and uncovered an age-dependent factor. No difference in SIVmac251 acquisition was observed in young or old naive controls (data not shown). Current Research and Future Plans 1. Define the epigenetic signatures of CD14+ monocytes, neutrophils, and Th2 CCR5negative T cells and study the miRNA signatures in extracellular vesicles (EV) that track with protective immunity. 2. Define the specificity and mechanistic intersection between protective vaccine-induced V2-targeted humoral responses and innate immunity 3. Development of the next generation of ALVAC based HIV vaccine candidate leveraging innate immunity to elicit durable protective immune profiles 4. Investigate whether monoclonal antibodies to V2 that inhibit a4B7 integrin binding protect against SIVmac251 acquisition, alone or in combination with innate monocyte memory immunity 5. Durability 6. Investigate the basis for the age bias vaccine efficacy and test adjuvants that may overcome it, such as IGF-1 and an alum-based liposome ALFA adjuvant with respect to innate immunity. 7: Design an HIV clade B based optimal DNA/ALVAC/gp120/alum vaccine regimen and test it in a phase I HIV vaccine trial in young volunteers.